Glycoconjugate vaccines comprising basic units of a molecular construct expressing built-in multiple epitopes for the formulation of a broad-spectrum vaccine against infections due to enteropathogenic bacteria

ABSTRACT

The present invention refers to new glycoconjugate antigens expressing built-in multiple epitopes and to polyvalent glycoconjugate vaccines intended for the protection of mammalians, and particularly for the protection of the human population from enteropathogenic bacteria, such as the Gram-positive anaerobic bacterium  Clostridium difficile  and the Gram-negative bacteria  Salmonella typhi, Escherichia coli, Vibrio cholerae, Shigella flexneri, Salmonella typhimurium, Salmonella enteritidis, Salmonella paratyphi  A,  Shigella sonnei, Shigella dysenteriae, Salmonella cholerasuis, Klebsiella, Enterobacter, Pseudomonas aeruginosa  and/or from viral gastrointestinal infections due to human noroviruses.

This application is a continuation of Ser. No. 15/329,205, filed Jan.25, 2017 which is a U.S. National Stage Application ofPCT/EP2015/066988, filed Jul. 24, 2015, which claims the priority ofItalian Patent Application Serial No. MI2014A001361, filed Jul. 25,2014.

The present invention refers to new glycoconjugate antigens expressingbuilt-in multiple epitopes and to polyvalent glycoconjugate vaccinesintended for the protection of mammalians, and particularly for theprotection of the human population from enteropathogenic bacteria, suchas the Gram-positive anaerobic bacterium Clostridium difficile and theGram-negative bacteria Salmonella typhi, Escherichia coli, Vibriocholerae, Shigella flexneri, Salmonella typhimurium, Salmonellaenteritidis, Salmonella paratyphi A, Shigella sonnei, Shigelladysenteriae, Salmonella cholerasuis, Klebsiella, Enterobacter,Pseudomonas aeruginosa and/or from viral gastrointestinal infections dueto human noroviruses.

Clostridium difficile is a spore-forming Gram-positive bacillusproducing two Exotoxins (Enterotoxin A and Cytotoxin B) which arepathogenic to humans.

C. difficile is the primary cause of antibiotic related infectiousdiarrhoea in elderly hospitalized patients in developed countries (Simoret al., 2002). Symptoms of C. difficile associated disease (CDAD) rangefrom diarrhoea to severe colitis, toxic megacolon, sepsis and death.Over recent years, increases in disease incidence, severity andrecurrence are largely due to the emergence of hypervirulent strainsassociated with epidemic hospital outbreaks combined with an increase inresistance to commonly used antibiotics (Rupnik et al., 2009).

A prophylactic vaccine capable of neutralizing the C. difficileEnterotoxin A and Cytotoxin B, the two Toxins of the pathogen, isreported to be as the candidate example of vaccine under industrialdevelopment (Donald R. et al., 2013).

Toxins A and B are very large proteins of 308 kDa and 270 kDa,respectively, that are structurally related, sharing homologousfunctional domains that mediate intracellular uptake and delivery of acytotoxic glucosyltransferase.

Toxin A (Enterotoxin) is composed of 2,710 AA and displays in itssequence 223 Lys residues (8.22% cationicity); Toxin B (Cytotoxin) iscomposed of 2,366 AA and displays in its sequence 156 Lys residues(6.59% cationicity).

Although these two toxins differ individually in their potency andeffects in “in vivo” models, past studies in animal models suggest thatthey both contribute to disease in natural infections (Lyerly et al.,1985). Furthermore, vaccination with both Toxin A and Toxin B—but notwith either alone—conferred protection in a hamster model of infection(Libby J. M. et al., 1982).

Recognition of the ability of the humoral immune response to controlCDAD prompted the successful use of passive immunotherapy with pooledhuman immunoglobulin containing anti-Toxin A and B antibodies to treatsevere CDAD (Salcedo J. et al., 1997). Furthermore, reduction inrecurrence of CDAD was achieved in a Phase I clinical trial with A and Banti-Toxin monoclonal antibodies in combination with standard antibiotictherapy (Lowy I. et al., 2010).

In addition, in a small study with three patients with chronic relapsingCDAD, an investigational vaccine using formalin-inactivated A and BToxoid antigens prevented CDAD recurrence (Sougioultzis S. et al.,2005).

Collectively, these observations provide validation for, and encouragefurther development of C. difficile Toxin A-based and Toxin B-basedvaccines to prevent CDAD. As above recalled, there are now two candidatevaccines in clinical trials, which are based on the tworecombinant/formalin-treated Toxoid proteins A and B.

Strategies for developing vaccines based on single specificities for C.difficile Toxoids (either detoxified by formalin treatment or by DNArecombinant technology) are well documented, as above recalled. Alsowell documented are the studies for using C. difficile recombinantenterotoxin A (rARU) as carrier protein for each of the capsular Ps ofS. flexneri type 2a, E. coli K1 and Pneumococcus type 14 (Pavliakova D.et al., 2000) prepared as single conjugates. Clearly, the simultaneousadministration of the single three conjugates inevitably results in anoverload for the immune system of the host due to the total, other thanheterogeneous, amount of injected carrier protein, namely therecombinant repeating unit of Clostridium difficile enterotoxin A(respectively 1.29 μg, 3.9 μg and 8.08 μg of rARU for each conjugatePn14-rARU, SF-rARU and K1-rARU).

Very recently, structural parts of the two Toxins have been used asnon-toxic carriers for the Ps II antigen of C. difficile (Romano M. etal, 2014). Although C. difficile also produces three different capsularPs, evidence is pointing in the direction of the two Toxins as targetfor efficaciously fighting the pathology, as in the historical cases ofDiphtheria and Tetanus infections.

None of these previous works, however, have reported on the possibilityto prepare a broad-spectrum enteric vaccine for inducing immunityagainst several carbohydrate antigens from antibiotic-resistantenteropathogenic bacteria (multiple-specificities) in a human host,particularly in a child, while using the minimum amount of carrierproteins for reducing the antigenic burden of the vaccine(s) on the hostimmune system, whilst maintaining the specific immunogenic activity andin vivo protection qualitatively achievable by administering monovalentconjugates. However, animal models do not allow to draw conclusions onthe quantitative aspects of the induced antibody titers by the multipleantigens of the invention, in comparison to the monovalent ones, sinceit is well known to the experts in the Field that only human infants canreliably discriminate among the eventually different helper T-dependentactivity of different models of conjugate entities.

The author of the present invention has now obtained multiple-epitopemolecular constructs as basic unit for the preparation of amultiple-epitopes glycoconjugate vaccine to be used as broad-spectrumenteric vaccine for the protection of the human population fromenteropathogenic bacteria. In fact, the author of the present inventionfocuses on the urgent problem nowadays reported for several intestinalpathogens which have become antibiotic resistant: the Gram-positiveanaerobic bacterium Clostridium difficile and the Gram-negative bacteriaSalmonella typhi, Escherichia coli, Vibrio cholerae, Shigella flexneri,Salmonella typhimurium, Salmonella enteritidis, Salmonella paratyphi A,Shigella sonnei Shigella dysenteriae, Salmonella cholerasuis,Klebsiella, Enterobacter, Pseudomonas aeruginosa. Because of theirincreasing antibiotic resistance, intestinal infections due to thispanel of bacteria may often lead to sepsis with consequent death of thehost.

Therefore, it is an object of the present invention an antigenicmultivalent molecular construct consisting of basic units comprising thehelper-T dependent carrier detoxified proteins selected betweenEnterotoxoid A and Cytotoxoid B from Clostridium difficile covalentlybound to a minimum of three carbohydrate structures fromenteropathogenic bacteria selected between bacterial polysaccharides ordetoxified lipopolysaccharides (such as SAEP-detoxified LPS orEndotoxoids) of different serological specificity, wherein eachcarbohydrate structure comprises at least one of the repeating basicepitopes consisting of a minimum of five to twelve monosaccharideresidues (preferably a minimum of eight to twelve monosaccharideresidues), wherein at least one mole of carrier protein is covalentlybound to at least one mole of type-specific or group-specificcarbohydrate structures, or to the total amount of carbohydratestructures being considered as the sum of the at least threetype-specific or group-specific carbohydrates. Preferably, saidsaccharide residues are assessed by molecular mass determination and NMRspectroscopy, said repeating basic epitopes being antigenically assessedby reactivity with type-specific or group-specific polyclonal ormonoclonal antibodies through the determination of their respectiveMIC50 values in the inhibition of their homologousPolysaccharide-Antibody reference system.

Enteropathogenic bacteria according to the present invention are thoseintestinal pathogens which have become antibiotic resistant such as: theGram-positive anaerobic bacterium Clostridium difficile and theGram-negative bacteria Salmonella typhi, Escherichia coli, Vibriocholerae, Shigella flexneri, Salmonella typhimurium, Salmonellaenteritidis, Salmonella paratyphi A, Shigella sonnei, Shigelladysenteriae, Salmonella cholerasuis, Klebsiella, Enterobacter,Pseudomonas aeruginosa.

According to a preferred embodiment of the present invention the toxoidproteins Enterotoxoid A and Cytotoxoid B from Clostridium difficile aredetoxified by chemical method, such as formalin-treatment, likehistorically known for diphtheria and tetanus toxoids, or by DNArecombinant technology.

In the molecular constructs according to the present invention each ofthe two toxoid proteins may support a minimum of three polysaccharidesof different antigenicity (such as oligosaccharides or polysaccharidesderiving from bacterial capsular polysaccharides) or a minimum of threedetoxified lipopolysaccharides (or LPS, Endotoxin) of differentantigenicity.

The molecular constructs obtained in this way with LPS, however, resultto be toxic because the Lipid A moiety of LPS is actively present in themolecular structure and can activate, via interaction with the CD14 andTLR4-like receptors, the pro-inflammatory cytokine cascade typical ofLPS. In order to pursue and achieve the safe use of the Toxoid-LPSconjugate entity, the LPS structure must therefore undergodetoxification.

This can be achieved by:

1) cleaving out the Lipid A moiety, or

2) by saturation of the Lipid A-binding site through a specific strategythat use the Synthetic Anti-Endotoxin Peptides (SAEP) in order to obtainEndotoxoids (alternatively named SAEP-detoxified LPS, SAEP-detoxifiedendotoxin) which conserve their complete supra-molecular antigenicrepertoire in the form of micelle-like structures (WO 2004/052394 A1).

The latter detoxification process is the preferred embodiment in thecontext of the present invention. Specifically, an Endotoxoid,originating from a given species-specific (immunotype) Endotoxin(Lipopolysaccharide) is prepared according to the scientific conceptreported by Rustici et al. (Science 259: 361-365, 1993) and in thepreviously disclosed molecular details reported in the U.S. Pat. No.6,951,652 and in the U.S. Pat. No. 7,507,718.

Therefore, an Endotoxoid is a molecular entity composed of an equimolarcomplex of SAEP, Synthetic Anti Endotoxin Peptides, and LPS (Endotoxin),which, in the form of a multiple-epitope conjugate with a C. difficileToxoid (A or B) satisfies the chemical equation:Toxoid-(LPS)₃+3SAEP→Toxoid-(Endotoxoid)₃(see also below Example 3).

According to preferred embodiment of the molecular constructs of thepresent invention, capsular polysaccharide antigens may be selectedbetween the group comprising Escherichia coli K types (1,2,5,12,13),Salmonella typhi (Vi antigen), Vibrio cholerae 0139 and Clostridiumdifficile.

Clostridium difficile, as a Gram-positive bacterium, also features acarbohydrate capsule involving at least three different Ps structures(PsI, PsII and PsIII).

According to an alternative embodiment of the molecular construct of thepresent invention, the two toxoid proteins serve as helper T-dependentcarriers for glycoconjugates of the detoxified lipopolysaccharides(preferably SAEP-detoxified LPS or Endotoxoid) specific for Shigellaflexneri 2a, Vibrio cholerae 01, Salmonella cholerasuis, Escherichiacoli 0157/101/111, Salmonella typhimurium, Salmonella enteritidis,Salmonella paratyphi A, Shigella sonnei, Shigella dysenteriae type 1 andSalmonella cholerasuis.

The molecular constructs according to the invention induce serologicalspecificity to the two carrier proteins (Enterotoxoid A and Cytotoxoid Bof C. difficile) and to each of the at least three carried carbohydratestructures (briefly denominated either Ps or LPS/Endotoxoid) bound toeach of the two carrier proteins, so that the relative specificantibodies exhibit neutralizing activity for the homologous naturaltoxins (Enterotoxin A and Cytotoxin B) of Clostridium difficile as wellas bactericidal activity for Salmonella typhi, Escherichia coli, Vibriocholera, Salmonella enteritidis, Salmonella paratyphi A, Shigelladysenteriae (other preferred carbohydrate antigens are from Shigellaflexneri, Salmonella typhimurium, Salmonella cholerasuis, Shigellasonnei and from C. difficile itself).

According to the above, and as a non limiting series of examples, theauthor has prepared the following molecular constructs:

-   -   Enterotoxoid A covalently bound to the Ps of S. typhi (Vi), V.        cholerae (0139) and E. coli (K1);    -   Cytotoxoid B covalently bound to the same three Ps of S. typhi        (Vi), V. cholerae (0139) and E. coli (K1);    -   Enterotoxoid A covalently bound to the LPS/Endotoxoids of S.        enteritidis, S. paratyphi A and S. dysenteriae;    -   Cytotoxoid B covalently bound to the same three LPS/Endotoxoids        of S. enteritidis, S. paratyphi A and S. dysenteriae.

The invention further relates to the above antigenic multivalentmolecular construct for use in a vaccine for the protection of a subjectfrom the infections due to at least one enteropathogenic bacteriaselected from Clostridium difficile, Salmonella typhi, Escherichia coli,Vibrio cholerae, Salmonella enteritidis, Shigella flexneri, Salmonellaparatyphi A, Salmonella dysenteriae, Salmonella cholerasuis or acombination thereof.

In a preferred embodiment either a single or a combination of differentantigenic multivalent molecular constructs may be used for thepreparation of the vaccine.

It is a further object of the present invention a vaccine formulationcomprising at least one antigenic multivalent molecular construct asabove in a physiologically acceptable vehicle, optionally together withan adjuvant or excipients pharmaceutically acceptable.

The antigenic molecular constructs may have an homogeneous or mixedpattern of carrier antigen and carried antigens. The term carrierantigen refers to the toxoid proteins Enterotoxoid A or Cytotoxoid Bfrom C. difficile; the term carried antigens refers to the carbohydratestructures (briefly denominated either capsular Ps or LPS/Endotoxoid)bound to each of the two carrier proteins. The term homogeneous or mixedrefer to the source of the carried antigens in respect to the carrierantigen (i.e. all the carrier and carried antigens originate from C.difficile; the carried antigens originate from the same or differentintestinal pathogens).

According to a preferred embodiment of the vaccine formulation of theinvention, the dose of each carrier antigen and/or carried antigensranges between 0.1 to 100 μg, preferably being 1-10 μg.

Preferably, said vaccine formulations further comprises a mineral or achemically synthetic or a biological adjuvant. Mineral or chemicallysynthetic or biological adjuvants can be used with the molecularconstruct disclosed in this application, in order to benefit from anyimmunological boost that can be effective in lowering the optimalimmunogenic dose in humans so to further reduce the total amount ofcarrier protein. Particularly, preferred inorganic adjuvants in thevaccine formulations according to the invention for use in human beingsare selected between Aluminium Phosphate (AlPO₄) and AluminiumHydroxide; preferred organic adjuvants are selected from squalene-basedadjuvants such as MF59, QF 21, Addavax; preferred biological antigensare selected between the bacterial antigens monophosphoryl-lipid A,trehalose dicorynomycolate (Ribi's adjuvant).

In vaccine formulations for use in the veterinary field Freund'sadjuvant (complete or incomplete) is preferred. The dose of adjuvant mayrange between 0.1-1 mg/dose, preferably being 0.5 mg/dose.

More preferably, such formulation is suitable for the administration bysubcutaneous or intramuscular or intradermal or transcutaneous route.Conveniently, such administration may be carried out by conventionalsyringe injection or needle-free tools.

The vaccine formulations according to the invention may be administeredaccording to a protocol which requires single or multipleadministrations, according to the physician, pediatrician or veterinaryinstructions.

The invention further relates to a broad-spectrum polyvalent vaccineformulation as above defined for use in medical human or veterinaryfield for the protection of a subject from the infections due to atleast one enterobacterial pathogens selected among Clostridiumdifficile, Salmonella typhi, Escherichia coli, Vibrio cholerae,Salmonella enteritidis, Shigella flexneri, Salmonella paratyphi A,Salmonella dysenteriae, Salmonella cholerasuis or a combination thereof.Preferably, said subject to be treated belongs to the paediatric and tothe elderly population.

The actual formulation of such vaccine (e.g.: the species-specificity ofthe Gram-negative enteric bacteria from which Ps and LPS derive) maydepend from the regional epidemiology so that each triad of antigenicconjugates, although using always one or both of the two carrierproteins Enterotoxoid A and Cytotoxoid B from C. difficile, purposelywill carry specific Ps or LPS/Endotoxoid antigens according to theselected regional epidemiology.

In a particular embodiment of the present invention the vaccineformulation comprises at least two different antigenic molecularconstructs wherein each of the two proteins Enterotoxoid A andCytotoxoid B from C. difficile may serve as carrier protein for thethree polysaccharides (PsI, PsII and PsIII) of C. difficile so that thetwo combined triads of conjugated antigens will represent a specificvaccine limited to the infections of C. difficile where the antitoxicactivity induced by the two protein toxoids may be paralleled by thelocal and systemic anti-capsular activity resulting in the clearance ofthe bacterium by the host immune system.

Such single-triad molecular constructs have been also formulated ascombined multi-valent compositions containing both kind of antigenicmolecular models for achieving the broadest antigenic spectrum such as:

-   -   Enterotoxoid A covalently bound to the Ps of S. typhi (Vi), V.        cholerae (0139) and E. coli (K1) combined with Cytotoxoid B        covalently bound to the same three Ps antigens;    -   Enterotoxoid A or Cytotoxoid B covalently bound to the Ps of S.        typhi (Vi), V. cholerae (0139) and E. coli (K1) combined with        Cytotoxoid B or Enterotoxoid A covalently bound to the three        Endotoxoid antigens of S. enteritidis, S. paratyphi A and S.        dysenteriae.

It has been recently reported on the experimental evidence that humanand mouse noroviruses infect B cells in vitro, and likely in vivo,through the involvement of enteric bacteria working as a stimulatoryfactor for norovirus infection. This biological synergism has beensuggested to be at the basis of the mechanism by which noroviruses maybecome infective and develop epidemic and sporadic gastroenteritis inhumans (Jones M. K. et al., Science, 346: 755-759, 2014).

In line with these observations, murine hosts undergoing antibiotictreatment for depleting the intestinal microbiota, have shown asignificant reduction of mouse norovirus replication in the experimentsreported by the authors.

From this evidence, the author of the present Application derived theprinciple of targeting the continuously expanding world ofantibiotic-resistant enteropathogenic bacteria with the vaccinecompositions herein disclosed in order to possibly limit, in parallel toenteric bacterial infections, the replication of noroviruses responsiblefor acute gastroenteritis.

Norovirus gastroenteritis is a widespread and potentially severe illnesscharacterized by the acute onset of nausea, vomiting, abdominal cramps,diarrhea and occasionally fever. Noroviruses are highly infective andeasily transmitted from person to person or via contaminatedenvironments. Epidemic outbreaks occur in community environments,particularly hospitals, hotels, schools, day care facilities and nursinghomes, with mounting socioeconomic cost to families, the health caresystem and businesses. Military units are significantly affected whenthe virus strikes, as outbreaks impact combat readiness. Severe clinicaloutcomes are reported in older adults, children and immunocompromisedindividuals in whom infection can lead to substantial complications andcan even lead to death. It is estimated that, worldwide, norovirusescause one in five cases of viral gastroenteritis. An estimated annual300 million cases of norovirus infection contribute to roughly 260,000deaths, mostly in low-income countries. Noroviruses are classified in atleast 5 genogroups and in at least 40 genotypes; their distribution inselected geographic areas has been recently evaluated in children andelders, with an incidence of 1,475 cases/100,000 persons-year in youngchildren (≤5 ys.) and 585 cases/100,000 persons-year in elders (≥65 ys.)(Chan M. et al, Scientific Reports, 2015). Over time, noroviruses evadenatural immunity by antigenic drift, which allows them to escape fromantibodies produced in response to earlier infections.

It is therefore another aspect of the present invention the provision ofbroad-spectrum polyvalent vaccine formulation for use in the preventionand/or treatment of enteropathogenic bacteria which then may target, inparallel, viral gastrointestinal infections due to human noroviruses.

Recent efforts to develop a norovirus vaccine have focused on virus-likeparticles (VLPs), which are constructed from molecules of the virus'scapsid (outer shell). In a phase I clinical trial, one multivalent VLPvaccine elicited antibody generation, but did not confer immunity to thetested strain of virus. However, in a more recent study, Lindesmith andcolleagues (2015) characterized serum specimens from ten multivalent VLPvaccine clinical trial participants for antibodies to vaccine VLPs andalso to VLPs representing viruses that were not contained in thevaccine. The researchers found that VLP vaccine can rapidly elicitantibody responses to a broad range of vaccine and non-vaccine VLPs,including to two VLPs representing human noroviruses that they could nothave previously encountered. Overall, antibodies to norovirus strains towhich participants had previously been exposed, dominated the immuneresponse. These findings may encourage the development of anorovirus-based vaccine assuming that this approach may overcome theability of noroviruses to evade immunity by antigenic drift. In anyevent, this would be a strategy directed to eventually contain the virusduring the phase of the infection in which the virus particles arespreading out of the bacterial cells hosting it, rather than to blockthe virus replication at the base, once it is still inside theenteropathogenic bacteria which are shielding it, as the author of thepresent Application is proposing by the use of a broad-spectrum vaccinetargeting enteropathogenic bacteria. Eventually, the concomitant and/orparallel use of these two strategies (e.g.: the use of the two vaccinestargeting the norovirus as well as its bacterial host) could constitutea powerful tool for achieving a broad-spectrum anti-viral protection forthe human host.

The present invention further relates to a conjugation process forpreparing the antigenic multivalent molecular construct according to theinvention (which employs the same chemistry disclosed in the patent EP1501542), wherein each of the at least three carbohydrate structuresselected among:

-   -   capsular polysaccharides of Salmonella typhi, Vibrio cholerae,        Clostridium difficile and Escherichia coli or    -   lipopolysaccharides from Clostridium difficile, Salmonella        typhi, Escherichia coli, Vibrio cholerae, Salmonella        enteritidis, Shigella flexneri, Salmonella paratyphi A,        Salmonella dysenteriae, Salmonella cholerasuis        is chemically activated to mono-functionality or        polyfunctionality by O-de-hydrogen uncoupling via oxidation and        reductive amination forming imine reduced bonds with an alkyl        diamine spacer, then derivatized to active esters, such        ester-derivative carbohydrate structures being finally and        simultaneously coupled to the amino groups of the polyfunctional        carrier protein Cytotoxoid B or Enterotoxoid A from C. difficile        through the formation of amide bonds;        wherein at least one mole of carrier protein is reacted with at        least one mole of carbohydrate structures, considering such a        total amount as the one composed by the molar sum of each of the        at least three type-specific or group-specific carbohydrate        structures. Preferably, said carbohydrate structures are        chemically activated in their corresponding diamine butyric acid        derivatives and the active esters are succinimidyl esters.

As an example, the chemical activation of the triad of polysaccharidefrom the capsule of S. typhi, E. coli and V. cholerae to theirhomologous Ps-DAB (diamine butyric acid derivative) has been performedaccording to the process disclosed by the Applicant in Claim 1 of EP1501542, while the polyfunctional carrier proteins were the EnterotoxoidA and Cytotoxoid B from C. difficile.

Alternatively, the conjugation process for preparing the antigenicmultivalent molecular constructs of the invention employs the chemistrydisclosed in Claim 8 of EP 1501542 involving simultaneous coupling (orstep-by-step coupling) of the amino groups of the poly-functionalcarrier proteins Cytotoxoid B or Enterotoxoid A from C. difficile withthe at least three different carbohydrate structures selected between

-   -   capsular polysaccharides of Salmonella typhi, Vibrio cholerae,        Clostridium difficile and Escherichia coli or    -   lipopolysaccharides from Salmonella typhi, Escherichia coli,        Vibrio cholerae, Salmonella enteritidis, Shigella flexneri,        Salmonella paratyphi A, Salmonella dysenteriae, Salmonella        cholerasuis        via reductive amination forming imine-reduced bond, such        carbohydrate structures being previously activated to        monofunctionality or polyfunctionality, with or without spacers,        by O-de-hydrogen uncoupling via oxidation;        wherein at least one mole of carrier protein is reacted with at        least one mole of carbohydrate structures, considering such a        total amount as the one composed by the molar sum of each of the        at least three type-specific or group-specific carbohydrate        structures.

According to the present invention the term mole referred to both thecarrier protein and the specific carbohydrate antigens encompasses thegeneral measure unit (a mole) or a fraction of it (i.e. micromole ornanomole or picomole, all representative for a fraction of it).

When the conjugation process according to the invention contemplatelipopolysaccharides, these should be detoxified. Therefore, theconjugation process further comprises an additional step ofdetoxification of said lipopolysaccharides alternatively by a) cleavingout the Lipid A moiety before or after the coupling reaction isperformed, or b) saturation of the Lipid A-binding site through aspecific strategy that use the Synthetic Anti-Endotoxin Peptides (SAEP,like the SAEP2 see Rustici et al., Science 259: 361-365, 1993) before orafter the coupling reaction is performed.

Preferably, such detoxified lipopolysaccharides are obtained through thelatter procedure disclosed by the same author in the U.S. Pat. No.6,951,652 (see page 16 and Claim 1) and U.S. Pat. No. 7,507,718 (seepages 33-34 and Claim 17) in order to obtain the correspondingEndotoxoids retaining the optimal antigenic features of thesupramolecular, micelle-like, LPS structure(s) for the optimalexpression of the relative immunogenic properties.

In addition to the above methods of detoxification, other methods may beused for the purpose and, among others, one may consider LPSdetoxification by genetic engineering through the modification of theenzymatic path leading to the synthesis of Lipid A as well asdetoxification by enzymatic or chemical hydrolysis of the ester-linkedfatty acid chains present in the Lipid A structure.

Furthermore, in a preferred embodiment of the conjugation process of theinvention, the carbohydrate structures of step a) comprise at least oneof the repeating basic epitopes consisting of a minimum of five totwelve monosaccharide residues as assessed by molecular massdetermination and NMR spectroscopy, said repeating basic epitopes beingantigenically assessed by reactivity with type-specific orgroup-specific polyclonal or monoclonal antibodies through thedetermination of their respective MIC50 values in the inhibition oftheir homologous

Polysaccharide-Antibody Reference System.

It represents a final object of the present invention an antigenicmultivalent molecular construct obtainable by the conjugation processabove outlined.

As it can be inferred, the above disclosed molecular model can befurther developed to contain more than three (for example four or five)different carbohydrate structures per single mole (or fractions of it)of protein carrier, this possibility depending from three mainparameters of the molecular construct:

a) the physical-chemical features of the carrier protein, whichstructure should feature the highest possible amount of Lysine residues(source of reactive —NH₂ groups);

b) the “ad hoc” selected polydisperse MW of the different carbohydratestructures featuring an optimal activation rate while limiting thenegative effects of steric hindrance phenomena in the coupling reaction,and

c) the efficiency of the chemistry used for the activation of thedifferent carbohydrate structures and for the synthesis of the molecularconstruct (the preferred chemistry for a high efficiency in the optimalactivation of carbohydrate structures is the O-de-hydrogen uncouplingvia oxidation, with or without spacer, while that for a high efficiencyin the conjugation reaction is through amide bond formation via activeesters between the carbohydrate structures and the carrier protein; alsopreferred for the conjugation reaction, is the chemistry which uses theformation of an imine reduced bond between the O-de-hydrogen uncouplingoxidized carbohydrate structures, with or without spacers, and thecarrier protein, via direct reductive amination).

The process of conjugation employed according to the invention foreseesthe multi-step activation of the (at least three) Ps or LPS (thatconsequently may have indifferently, although homogeneously, either lowor high MW) in order to optimize the coupling yields with the carrierprotein.

The stoichiometric features of the present molecular constructs (w/wratio Protein/Ps or Protein/LPS), which are in turn related to theimmunizing dose of the molecular constructs have been carried out by theimmunochemical method disclosed in the international patent applicationNo. PCT/EP2014/051670.

This has allowed the possibility in the present invention to determinethe quantitative amount of Ps or LPS even when having very similarstructures if present in the same molecular construct.

Finally, the present invention is directed to limit the amount ofcarrier protein in the vaccine formulation to the minimumimmunogenically possible as related to the broader antigenic repertoireof the conjugate antigens, in order to contain the antigenic burden onthe host's immune system for the molecular constructs obtainable throughthe conjugation processes above disclosed. This strategy is coherentwith the containment of the clinical phenomenon today known as“carrier-specific immune interference” which is related to the amount ofcarrier protein used in a given glycoconjugate vaccine composition whenconsidering the context of other vaccines administered during theimmunization path of the mammalian host (Dagan R. et al, 2010; Lee L. H.and Blake M. S., 2012).

In the following experimental section the invention will be disclosed inmore detail according to preferred embodiments. Such embodiments shouldbe considered not limitative for the scope of protection of the presentpatent disclosure, but merely for illustrative purpose.

EXAMPLES Example 1

i) Synthesis of the Tetravalent Conjugate Antigen ComprisingPolysaccharides of S. typhi(Vi), E. coli (K1) and V. cholerae (0139)with the Carrier Protein Enterotoxoid A;

ii) Synthesis of the Tetravalent Conjugate Antigen ComprisingPolysaccharides of S. typhi (Vi), E. coli (K1) and V. cholerae (0139)with the Carrier Protein Cytotoxoid B.

Chemical Activation of the Three Ps to the Homologous Ps-DAB (DiamineButyric Acid Derivative)

This step has been performed according to the process disclosed by theApplicant in the Claim 1 (step A1) of the above mentioned patentEP1501542. Specific controls of such activation as well as the obtainedcharacteristics of the activate Ps structures has been performed using¹H-NMR spectroscopy as reported in the international application No.PCT/EP2014/051670.

¹H-NMR Analysis of Ps-DAB Derivatives

1. Solution of Ps and Ps-DAB Derivatives for NMR Analysis

3-4 mg of polysaccharide sample (PS) or PS-DAB is solved in 0.7 ml ofD₂O—phosphate buffer and transferred into a 5 mm NMR tube. Theconcentration of phosphate buffer prepared in D₂O is 100 mM, pH=7.Trimethylsilylpropionic acid sodium salt (TSPA), (CH₃)₃Si(CD₂)₂COONa isused as an internal reference. The concentration of TSPA is 1 mM.

2. NMR Equipment

High field NMR spectrometer (600 MHz) is used. A high resolution 5 mmprobehead with z-gradient coil capable of producing gradients in thez-direction (parallel to the magnetic field) with a strength of at least55 G·cm⁻¹ is employed.

3. Setup of NMR Experiments

After the introduction of the sample inside the magnet all the routineprocedures have been carried out: tuning and matching, shimming, 90degree pulse calibration. Presaturation can be used to suppress theresidual HDO signal. For good presaturation the centre of the spectrum(O1) must be set exactly on the HDO signal (about 4.80 ppm), and goodshimming is desirable as well.

After adjustment of parameters for presaturation, the parameters ofdiffusion gradient experiments are checked. The stimulated echo pulsesequence using bipolar gradients with a longitudinal eddy current delayis used.

4. Fingerprinting of DAB-Activation

Group —CH₂—NH₂ at 3.08 ppm

Group —CH₂—NH—CH₂— at 3.17 ppm

5. % of DAB Activation on Ps

% of DAB activation is in the range value of 0.5-5.0% moles DAB/molesBRU (Basic Repeating Unit of the Group-specific Ps) with an optimalmolar range 1.5-3.0%.

Derivatization of Ps Vi, Ps 0139, Ps K1 to their Homologous ActiveEsters as Ps-DAB-MSE Derivatives

This step has been performed according to the process disclosed by theapplicant in Claim 8 of the European Patent EP 1501542, herewithincluded as a reference.

Simultaneous Coupling of the Three Activated (Poly-Functional) Ps to the(Poly-Functional) Carrier Protein Enterotoxoid A or Cytotoxoid B

The chemical synthesis of the conjugate, also known as couplingreaction, has been performed according to the process disclosed by theapplicant in Claim 8 of the European Patent EP1501542. The procedure,however, can be here considered as innovative because the three couplingreactions are simultaneously run, rather than proceeding in one couplingreaction at the time (or step-by-step process).

This procedure may be preferred to the step-by-step coupling of eachPs-activated antigen for the simple reason of shorting the reactiontime, therefore improving the efficiency of the reaction, provided thatthe three activated-Ps are in the condition to comparatively compete atthe equilibrium for the coupling reaction (this feature includecomparable average MW, comparable range of Ps-DAB activation andcomparable stoichiometric ratios among the reacting groups of theprotein and those of the activated Ps).

The appropriate stoichiometry of reaction keeps in consideration thetotal amount of succinimidyl esters relative to the three Ps antigensactivated and the amino groups of the carrier protein available.Stoichiometry is preferentially set as to consider the reactivity of nomore than 20-25% of the amino groups available in the structure ofEnterotoxoid A or Cytotoxoid B (as an example) in order for the proteinto optimally conserve its antigenic repertoire.

The coupling reaction of Enterotoxoid A or Cytotoxoid B (brieflyindicated as Toxoid) with Ps-DAB derivatives (Ps-DAB) is consistent withthe following stoichiometry:Toxoid+4Ps-DAB(MSE)→Toxoid-(Ps)₃+Ps-DAB(MSE)

Where the entity Ps-DAB(MSE) derivatives refer to the total of equalparts of each of the three type-specific Ps structures in reactionyielding a conjugate averaging 1 mole of protein for the total of 3moles of type-specific Ps carried, plus the due excess of Ps-DAB(MSE)derivatives, as ruled by the equilibrium constant:

${{Keq} = {\frac{\left\lbrack {{Toxoid}\text{-}({Ps})_{3}} \right\rbrack\left\lbrack {{Ps}\text{-}{{DAB}({MSE})}} \right\rbrack}{{\lbrack{Toxoid}\rbrack\left\lbrack {{Ps}\text{-}{{DAB}({MSE})}} \right\rbrack}^{4}} = \frac{\left\lbrack {{Toxoid}\text{-}({Ps})_{3}} \right\rbrack}{{\lbrack{Toxoid}\rbrack\left\lbrack {{Ps}\text{-}{{DAB}({MSE})}} \right\rbrack}^{3}}}}\quad$

The equation refers to the concentration of the total active esters(MSE) deriving from the sum of equal parts of the DAB-activated Psantigens, which are in turn comparable to the amount of the DAB linkerquantitated by ¹H-NMR spectrometry which is present in each activated Psantigen (conversion rate of Ps-DAB to Ps-DAB(MSE)≥98% on molar basis).

The chemical equation makes evidence for the complete glycosylation ofthe Toxoid carrier protein. The equation also shows that the conjugationreaction depends from the concentrations of both reagents, thenucleophile (Toxoid through the epsilon-NH₂ groups of its Lys residues)and the electrophile (the carbonyl moiety of the ester groups of Psderivatives) therefore being defined as S_(N)2 reaction.

The above considerations are consistent with the experimentalobservation that the highest yield in the glycosylation reactionobtained with Toxoid as carrier protein has been 100% of the carrierprotein and about 80% (w/w) of the Ps-DAB-derivatives present inreaction, with the remaining part of them being a low amount ofuncoupled Ps-derivatives necessary for pushing to the right side theequilibrium.

In this type of reactions, the solvent affects the rate of reactionbecause solvents may or may not surround the nucleophile, thus hinderingor not hindering its approach to the carbon atom. Polar aproticsolvents, are generally better solvents for this reaction than polarprotic solvents because polar protic solvents will be solvated by thesolvent's hydrogen bonding for the nucleophile and thus hindering itfrom attacking the carbon with the leaving group. A polar aproticsolvent with low dielectric constant or a hindered dipole end, willfavor S_(N)2 manner of nucleophylic substitution reaction (preferredexamples are: DMSO, DMF, tetrahydrofuran etc.).

The temperature of reaction, which affects K_(eq), is the lowestcompatible with the use of the solvent chosen, when considering that thereaction is a spontaneous one (therefore being exothermic) and thereforeis generally set between a temperature of 4° and 20° C.

In addition to the conjugation chemistry above detailed, otherchemistries can be used to achieve the synthesis of the multivalentconjugate antigen; among these, the direct coupling of the protein (viareductive amination) to the oxidized Ps (via O-de-hydrogen uncoupling)or the use of heterologous and chemically complementary linkers that mayserve to activate the Ps and the protein.

Also, in addition to the strategy of using chemistries leading to obtainmultivalent cross-linked protein-Ps conjugates via thepoly-functionality of the protein and that of the Ps components, one mayconsider the synthesis of the presently disclosed antigenic multivalentmolecular construct as based on oligosaccharides derived from capsularPs or from Lipid A-deprived oligosaccharides of LPS, activated at theirend-reducing group for then being coupled to the carrier protein, as theapplicant showed in another model of conjugate antigen in the abovementioned paper Porro M. et al. in Molecular Immunology, 23: 385-391,1986.

Finally, the disclosed molecular construct might be thought to beprepared by enzymatic glycosylation in bacterial or yeast cells or otherengineered living cells, using “ad hoc” DNA-recombinant techniques.

Example 2

iii) Synthesis of the tetravalent conjugate antigen comprising LPS(Lipopolysaccharides) of S. enteritidis, S. paratyphi A and S.dysenteriae with the carrier protein Cytotoxoid B;

iv) Synthesis of the tetravalent conjugate antigen comprising LPS(Lipopolysaccharides) of S. enteritidis, S. paratyphi A and S.dysenteriae with the carrier protein Enterotoxoid A.

Chemical Activation of the Three LPS to the Homologous LPS-DABDerivatives (Diamine Butyric Acid Derivative)

The three LPS, were chemically derivatized in their O-antigencarbohydrate moiety to the corresponding -DAB derivatives (see the belowscheme showing the DAB-activated area within the O-antigen carbohydratemoiety which is the most hydrophilic part of the LPS molecule). The“core” structure is difficult to be activated because is very close tothe hydrophobic area (Lipid A), which is a quite kriptic structureresponsible for the micelle-like structure of LPS, which is alsoresponsible for the biological toxicity of LPS (e.g. local and systemicinflammation, TNF- and IL6-mediated, followed by pyrogenicity) as wellas for the optimal expression of antigenicity and immunogenicity. In apreferred embodiment of the present Application, the biological toxicityof LPS is then selectively blocked through the high affinity bindingwith SAEP, which preserves such optimal features of LPS linked to itssupramolecular, micelle-like, structure.

The step of DAB-activation has been performed according to the processdisclosed by the Applicant in the Claim 1 (step A1) of the abovementioned patent EP1501542. Specific controls of such activation as wellas the obtained characteristics of the activate Ps structures has beenperformed using ¹H-NMR spectroscopy as reported in the internationalpatent application No. PCT/EP2014/051670.

¹H-NMR analysis on the -DAB derivatives were conducted as above reportedfor Example 1.

The following scheme represents the general LPS structure ofEnterobacteriaceae with the located sites of DAB-activation (necessaryfor conjugation to the carrier protein) and the necessary biologicaldetoxification, preferentially performed by SAEP (Synthetic AntiEndotoxin Peptide), which allows to achieve detoxification while LPSretaining its supramolecular, micelle-like, antigenic structure).

Derivatization of LPS of S. enteritidis, S. paratyphi a and S.dysenteriae to their Homologous Active Esters as LPS-DAB-MSE Derivatives

This step has been performed according to the process disclosed by theapplicant in Claim 8 of the European Patent EP 1501542.

Simultaneous Coupling of the Three Activated (Poly-Functional) LPS tothe (Poly-Functional) Carrier Protein Enterotoxoid A

The chemical equation reported above in Example 1, also applies to theconjugates of Toxoids and LPS-derivatives:Toxoid+4LPS-DAB(MSE)→Toxoid-(LPS)₃+LPS-DAB(MSE)

So that:

${{Keq} = {\frac{\left\lbrack {{Toxoid}\text{-}({LPS})_{3}} \right\rbrack\left\lbrack {{LPS}\text{-}{{DAB}({MSE})}} \right\rbrack}{{\lbrack{Toxoid}\rbrack\left\lbrack {{LPS}\text{-}{{DAB}({MSE})}} \right\rbrack}^{4}} = \frac{\left\lbrack {{Toxoid}\text{-}({LPS})_{3}} \right\rbrack}{{\lbrack{Toxoid}\rbrack\left\lbrack {{LPS}\text{-}{{DAB}({MSE})}} \right\rbrack}^{3}}}}\quad$

The equation refers to the concentration of the total active esters(MSE) deriving from the sum of equal parts of the DAB-activated LPSantigens, which are in turn comparable to the amount of the DAB linkerquantitated by ¹H-NMR spectrometry which is present in each activatedLPS antigen (conversion rate of Ps-DAB to Ps-DAB(MSE)≥98% on molarbasis).

The chemical synthesis of the conjugate, also known as couplingreaction, has been performed according to the process disclosed by theapplicant in Claim 8 of the European Patent EP1501542. The procedure,however, can be here considered as innovative because the three couplingreactions are simultaneously run, rather than proceeding in one couplingreaction at the time (or step-by-step process). This procedure may bepreferred to the step-by-step coupling of each Ps-activated antigen forthe simple reason of shorting the reaction time, therefore improving theefficiency of the reaction, provided that the three activated-Ps are inthe condition to comparatively compete at the equilibrium for thecoupling reaction (this feature include comparable average MW,comparable range of LPS-DAB activation and comparable stoichiometricratios among the reacting groups of the protein and those of theactivated LPS). The molecular constructs obtained in this way, however,result to be toxic because the Lipid A moiety of LPS is actively presentin the molecular structure. In order to pursue and achieve the safe useof the Toxoid-LPS conjugate entity, the LPS structure must thereforeundergo detoxification alternatively through cleaving out the Lipid Amoiety, or by saturation of the Lipid A-binding site through a specificstrategy that use the Synthetic Anti-Endotoxin Peptides (SAEP). Thelatter is the preferred embodiment in the context of the presentinvention (see next example 3).

Example 3: Preparation of Enterotoxoid A-Endotoxoid Conjugates andCytotoxoid B-Endotoxoid Conjugates from their Homologous EnterotoxoidA-LPS (Endotoxin)/Cytotoxoid B-LPS (Endotoxin) Conjugates

Endotoxoids are non-toxic antigens able to induce specific immunologicalactivity against their homologous LPS which are the native main toxicantigens exposed on the surface of the Gram (−) bacteria.

A comprehensive, publically available, textbook which is exhaustive onthe many scientific aspects of Endotoxin antigens originating fromGram-negative bacteria is “Endotoxins” by Kevin L. Williams, Editor,Informa Health Care USA Inc., publisher, New York (2007).

An Endotoxoid is a molecular entity composed of an equimolar complex ofSAEP, Synthetic Anti Endotoxin Peptides, with the Lipid A moiety of LPS(Endotoxin):Toxoid-(LPS)₃+3SAEP→Toxoid-(Endotoxoid)₃

An Endotoxoid, originating from a given species-specific (immunotype),Endotoxin (Lipopolysaccharide), is prepared according to the scientificconcept reported by Rustici et al. (Science 259: 361-365, 1993) and inthe previously disclosed molecular details reported in the U.S. Pat. No.6,951,652 and in the U.S. Pat. No. 7,507,718.

The immunological activity of an Endotoxoid involves polyclonalantibodies of the IgG (mainly) and IgM isotypes having biologicalactivity (bactericidal effect) via the mechanism known in immunology asOpsonophagocytosis (OP, or antibody-mediated engulfing of bacteria inmacrophages and PMC) and Direct Bactericidal (DB, antibody-mediatedlysis of the bacterial cell wall), both mechanisms being mediated byactivation of the complement pathway.

Endotoxoids are helper-T dependent antigens in animal models but not yetexperienced in human infants, where the immune system is not fullydeveloped until an age over 2 years. For this reason, the conjugation tohelper-T dependent carrier proteins like the two above reported proteinToxoids of C. difficile has been considered in the present Applicationfor preparing the desired vaccine product.

Accordingly, the conjugates of Enterotoxoid A or Cytotoxoid B withselected species-specific LPS (Endotoxin) have been reacted with SAEP2(Rustici et al., Science 259: 361-365, 1993) in the conditions generallyreported in the U.S. Pat. No. 7,507,718 (see pages 33-34 and Claim 17),in order to achieve the detoxification of the Toxoid-conjugated LPS sothat the relative homologous Toxoid-conjugated Endotoxoids are formed.

The following Toxoid-conjugated Endotoxoids have been prepared:

-   -   Enterotoxoid A covalently conjugated to Endotoxoids of S.        paratyphi A, S. dysenteriae, S. enteritidis;    -   Cytotoxoid B covalently conjugated to Endotoxoids of S.        paratyphi A, S. dysenteriae, S. enteritidis;

CRM197 covalently conjugated to Endotoxoids of S. paratyphi A, S.dysenteriae, S. enteritidis as a well established helper-T dependentcarrier protein useful in controlling the immunization experiments inanimal models.

Example 4: Combination of the Tetravalent Conjugate Antigen ComprisingPolysaccharides of S. typhi (Vi), E. coli (K1) and V. cholerae (0139)Conjugated to the Carrier Protein Enterotoxoid A, with the TetravalentConjugate Antigen Comprising LPS/Endotoxoids of S. enteritidis, S.paratyphi A and S. dysenteriae Conjugated to the Carrier ProteinCytotoxoid B

The combination is prepared by associating the two kind of molecularmodels at the dose as appropriate for immunogenic studies in animalmodels below reported in the Example 8.

Example 5: Physical-Chemical Analysis of the Antigenic MultivalentMolecular Construct Comprising the Polysaccharides of S. typhi (Vi), E.coli (K1) and V. cholerae (0139) Conjugated to the Carrier ProteinEnterotoxoid A or Cytotoxoid B

The GPC analysis (Gel Permeation Chromatography) on Sepharose 4B-CL hasbeen used to perform the physical analysis of the antigenic multivalentmolecular construct of Example 1. Purification of the High MolecularWeight (HMW)-multivalent antigen is simply obtained by collecting andpooling the eluted fractions from Kd=0.00 to Kd=0.30.

Polymers of the basic unit of the molecular construct are obtained ascross-linked molecular entities because of the polyfunctionality of thePs antigens (about 2% of DAB activation, on molar basis, as evidenced by¹H-NMR spectroscopy) and the polyfunctionality of the carrier protein(ca. 104 reactive amino groups/mole Toxoid A, as determined by TNBSreaction, remaining from the native 223 Lys residues of the Toxin A+1amino terminal AA, within the structure encompassing the whole 2,710 AAof the sequence; ca. 85 reactive amino groups/mole Toxoid B, asdetermined by TNBS reaction, remaining from the native 156 Lys residuesof the Toxin B+1 amino terminal AA, within the structure encompassingthe whole 2,366 AA of the sequence).

In light of the above, the conjugate under analysis appears as apolydispersed, monomeric to polymeric, molecular entity which containsthe basic unit of the molecular construct reported in the chemicalequation, with a HMW which derives from the basic polymerized unitencompassing the Enterotoxoid A (MW=3.08×10⁵) or Cytotoxoid B(MW=2.70×10⁵) and an average of MW=10⁵ for each of the three Ps/LPSantigens (or a total of ca. 3.0×10⁵) resulting in a comprehensiveaverage MW of 6.10×10⁵ per basic unit; accordingly, the severalcross-linked units of such basic structure is reaching several millionsand are mainly eluted at the Vo of the Sepharose 4B-CL column.

The w/w ratio between the carrier protein and each of the threetype-specific Ps is ca. 3.6 (Table 1, below); this w/w ratio yields anaverage molar ratio (R) protein/type-specific Ps of ca. 1.0,corresponding to an average ratio of one mole of protein/mole oftype-specific Ps, as well suggested by the chemical equation.Accordingly, the experimentally obtained, cross-linked, molecular entityresponds to a molecular model constituted by several polymeric units ofthe basic unit just consisting of one mole of carrier protein carrying atotal of three moles of type-specific Ps (one mole for eachtype-specific Ps).

Example 6: Immunochemical Analysis of the Antigenic MultivalentMolecular Construct Enterotoxoid A-PsVi, PsK1, Ps0139 or CytotoxoidB-PsVi, PsK1, Ps0139

The GPC purified molecular construct was analyzed by inhibition-ELISAfor determining the serological specificity of the four serum differentpolyclonal antibodies (PAbs) and for determining the qualitative andquantitative presence of each antigen of the construct, as disclosed inthe international patent application PCT/EP2014/051670.

The comparison between chemical titration and immunochemical titrationof carbohydrate antigens for testing their quantitative equivalence, wasperformed by the use of inhibition-ELISA, through the experimentallydetermined parameter MIC₅₀ (Minimal Inhibitory Concentration of theselected carbohydrate antigen working as inhibitor of the homologousreference Ps-Ab reaction) in order to evaluate and correlate accuracyand precision of the immunochemical method with respect to the chemicalone in the analytical control of such a kind of molecular construct.

Example 7: Determination of the Concentration for the CarbohydrateAntigen in Either Activated or Multivalent Conjugated Form: Comparisonof Chemical Titration Vs. Immunochemical Titration

Immunochemical titers are obtained according to the method reportedabove relative to the Inhibition-ELISA as compared to chemical titersobtained according to the methods reported in the specific sections ofthe international patent application PCT/EP2014/051670; immunochemicaltiters of unknown samples of each of the three carbohydrate-specificantigens, either in activated or conjugated form, were determined byinterpolation on the linear part of a reference standard curve built byinhibition-ELISA using known, chemically titred, carbohydrate antigenamount.

The same methodology described for the qualitative and quantitativeimmunochemical analysis of each molecular construct above reported, isthen used for characterization of the final formulation of thepolyvalent vaccine containing the association of the two molecularconstructs, each constituted by a triad of Ps/LPS (Endotoxoid)conjugates of the two Toxoids of C. difficile used as carrier proteins,in order to get the complete characterization of an exemplificative4-valent or 8-valent vaccine.

Example 8: Vaccine Formulation as Related to the Stoichiometry of theMulti-Valent Molecular Constructs

Such kind of broad-spectrum formulations for an Enteric Vaccine can besafely prepared by the use of molecular constructs of the presentinvention, which allows a reduced use of protein carrier for carryingsuch a number of conjugated Ps and LPS (Endotoxoids) antigens. Asspecifically referred to an exemplified formulation of an EntericVaccine containing an 8-valent formulation which includes the mostprevalent, epidemiologically significant, specific Ps andLPS/(Endotoxoids), the following molecular constructs (Table 1) havebeen synthesized and analyzed as an extended exemplification of thepreferred embodiments, according to the methods reported above in thevarious Examples detailing the molecular constructs based onEnterotoxoid A and Cytotoxoid B carrying Ps/LPS (Endotoxoids), as wellas the combination of the two.

The total amount of the two carrier protein Toxoids exemplified in this8-valent Enteric Vaccine prepared and formulated according to theprocedures reported in this application and defined by the stoichiometryof the resulting molecular constructs, each one expressing built-inmultiple epitopes, is coherent with the following molar compositionrelatively to the dose of each molecular construct containing ca. 1 ugof each of the two carrier protein Toxoids (MW=308K and 270K,respectively) and ca. 0.3 μg of each of the three selectedDAB-activated, type-specific, Ps/LPS (Endotoxoid) antigens (averageMW=100K based on two different criteria of analysis, that is estimatingthe average sizing by molecular filtration on calibrated filtermembranes and estimating sizing by GPC, in all cases using referencecarbohydrate molecules like Dextrans of various MW).

TABLE 1 Molecular Average weight ratio Average molar ratio ConstructToxoid/Ps Toxoid/Ps EnteroTox A for: Ps_(E. coli) 3.30 1.08Ps_(S. typhi) 3.80 1.24 Ps_(V. cholerae) 4.05 1.33 CytoTox B for:EndoTox_(S. enteritidis) 3.65 1.36 EndoTox_(S. paratyphi A) 3.01 1.12EndoTox_(S. dysenteriae) 3.90 1.45

In the exemplified molecular constructs, the mean of the (w/w) ratioProtein to Ps/LPS is: 3.61±0.39 (10.8%) corresponding to the mean of the(mol/mol) ratio: 1.26±0.14 (11.1%).

The concept of calculating and comparing the features of conjugateantigens on molar basis is fundamental because the immune systemprocesses antigens on molar basis, as Nature does in each chemical orbiochemical reaction of transforming matter, therefore referring to theantigen's MW.

Accordingly, depending from the average MW of each type-specific Ps/LPSantigen and that of the protein carrier Toxoids, the molar ratios ofconjugate antigens are subject to change by the selection of theirantigen components. It is mostly preferred that molar ratios betweencarrier protein and each type-specific Ps antigen be equal to or higherthan 1.0 for a likely optimal expression of helper T-dependency. Inaddition to this molar parameter, it is also important considering theaverage amount of covalent bonds interposed between the protein and eachtype-specific carbohydrate antigen, which parallels the activation rateof the type-specific polysaccharide, since this hybrid molecular regionis the one experimentally suggested as responsible for the acquiredhelper T-dependent properties of a conjugate molecule (Arndt and Porro,1991).

It is however possible to synthesize the molecular constructs accordingto different stoichiometries of synthesis, as detailed in theinternational patent application PCT/EP2014/051670, by addressing theamount of reagents participating to the chemical equilibrium reported inthe above chemical equation, which may lead to a molecular construct ofdifferent stoichiometry, where the amount of helper T-dependent carrierprotein in the molecular construct can be optimally selected accordingto the optimal expression of immunogenicity of such molecular constructin the various age groups of the human population. In both, aboveexemplified, 4-valent to 8-valent formulations, containing one to twomolecular constructs each carrying three type-specific Ps/LPS, the totalamount of each carrier protein Toxoid is ca. 1 μg, while the conjugatedtype-specific Ps/LPS (Endotoxoid) are in the amount of ca. 0.3 μg,respectively.

Accordingly, it is the purpose of the above reported embodiments toprovide evidence of the fact that the disclosed multivalent antigenicmolecular construct with built-in epitopes can be synthesized in a broadrange of stoichiometric parameters in order to then properly define, inmammalian hosts and particularly in humans, the optimal dose of theconstruct even when considering the different age-groups (from infantsto elders) to be immunized by such a broad-spectrum vaccine formulation.

Table 2 below, shows different molecular models obtained for the aboveconcept, by making use of the same chemical reaction of synthesis,although using different “ad hoc” chosen stoichiometries for thereagents participating to the equilibrium.

Here below, are reported some considerations on the two Toxoids used inthe present application, Enterotoxoid A and Cytotoxoid B, since they are(or may be) chemically-treated derivatives of the homologous Toxins.This historic procedure, used for historic vaccines like Tetanus Toxoidand Diphtheria Toxoid, is necessary for having the Toxins purposelydetoxified for a safe human use as immunogens. In the presentApplication, we have considered the average MW of the purified Toxoidsas being comparable to that of the Toxins from which they derive.However, among other features, the marked difference between Toxoids andToxins resides in the amount of residual primary amino groups from theLysine residues which remain in the Toxoid structures after the chemicaldetoxification. An average of 47% to 54% reactive amino groups are aboutto be detected in the Toxoids with respect to those originally presentin the structure of the homologous Toxins, which work as nucleophylicgroups in the coupling reaction with the activated Ps/LPS antigens. Whencomparing the structure of the two Toxoids to that of a consolidated,historic, carrier protein like CRM197, in terms of capability to competein the coupling reaction as nucleophylic reagent, one may determine thatToxoid A has ca. 104 amino groups/mole (MW=3.08×10⁵ for 2,710 AA) whileToxoid B has ca. 85 amino groups/mole (MW=2.7×10⁵ for 2,366 AA), so thatthe molar density of them (which we define as “molar nucleophileactivity”) is 3.84% in Enterotoxoid A and 3.60% in Cytotoxoid B, twoparameters that are significantly lower than that calculated for CRM197(7.47%) which does have a higher capability to serve as nucleophylicreagent in a given coupling reaction (as detailed in the internationalpatent application No. PCT/EP2014/051670). However, given thesignificant difference in the MW of the two protein Toxoids (basically afactor=5.3 and 4.7 in their favor with respect to CRM197) the molarratios of the protein carrier, for each of the carried carbohydrateantigens selected in the molecular constructs, may result advantageousfor the Toxoids when one is willing to limit the amount of carrierprotein/dose in a polyvalent formulation. In fact, at comparable weightdoses of the two carrier protein Toxoids, they result to be about 5.0times lower than CRM197 on molar basis. Accordingly, attention must bepaid to the fact that the carrier MW is an important parameter affectingthe physical-chemical features of the conjugates and may limit thepossibility to obtain a molar ratio Toxoid/specific Ps/LPS with a value≥1.0 for the optimal induction of T-helper dependency in the host'simmune system.

Table 2 lists all the molecular models synthesized for the work detailedin the present Application, representative of the variousstoichiometries used for the purpose, which are dependent from: i) theMW of the carrier protein used; ii) the molar nucleophile activity ofsuch carrier proteins (expressing the amount of —NH₂ groups/mole ofprotein); iii) the average MW of the activated Ps/LPS antigens and, iv)the respective activation rate of the Ps/LPS antigens (DAB-MSE groupsfor then reacting with the —NH₂ groups of the protein). The exemplifiedmolecular models make evidence for the flexibility of the chemistryadopted and the fact that the carrier protein may be present in theconjugate entity in a broad variety of ponderal and molar ratios, above1.0 and below 1.0. In particular, the molar ratio Protein/Ps ranged fromat least 0.3 to 1.0 when considering each type-specific orgroup-specific Ps present in the glycoconjugate, and from at least 0.3to 1.0 when considering the total of the three Ps, each Ps contributingfor about one third to the total amount finally present in theglycoconjugate.

TABLE 2 Molecular Average weight ratio Average molar ratio ConstructToxoid/Ps Toxoid/Ps EnteroTox A for: Ps_(E. coli) 3.30 1.08Ps_(S. typhi) 3.80 1.24 Ps_(V. cholerae) 4.05 1.33 Ps_(E. coli) 1.050.34 PS_(S. typhi) 1.15 0.37 Ps_(V. cholerae) 1.03 0.33EndoTox_(S. enteritidis) 3.35 1.09 EndoTox_(S. paratyphi A) 3.00 0.97EndoTox_(S. dysenteriae) 3.20 1.04 EndoTox_(S. enteritidis) 1.13 0.37EndoTox_(S. paratyphi A) 1.20 0.39 EndoTox_(S. dysenteriae) 1.05 0.34CytoTox B for: EndoTox_(S. enteritidis) 3.65 1.36EndoTox_(S. paratyphi A) 3.01 1.12 EndoTox_(S. dysenteriae) 3.90 1.45EndoTox_(S. enteritidis) 1.23 0.46 EndoTox_(S. paratyphi A) 1.02 0.38EndoTox_(S. dysenteriae) 1.15 0.43 Ps_(E. coli) 3.60 1.33 Ps_(S. typhi)3.45 1.28 Ps_(V. cholerae) 3.85 1.43 Ps_(E. coli) 1.25 0.46Ps_(S. typhi) 1.10 0.40 Ps_(V. cholerae) 1.43 0.53

Example 9: Immunological Analysis in Animal Models of the AntigenicMultivalent Molecular Constructs of Enterotoxoid A and Cytotoxoid B(Originating from the Homologous Toxins of C. difficile) CarryingPolysaccharides (S. typhi, V. cholerae, E. coli) or LPS/Endotoxoids (S.paratyphi A, S. dysenteriae, S. enteritidis)

The two kind of conjugates using the two protein Toxoids from C.difficile, have been experienced in a murine animal model for activeimmunization experiments. As helper-T dependent control immunogen, thehomologous conjugates of CRM197 were used in parallel experiments.

Vaccine Formulation for Ps-Conjugates

Enterotoxoid A and Cytotoxoid B conjugates of PsVi, Ps0139 and PsK1 werecombined. Stoichiometric features of the conjugates showed a mean ratioProtein/each of the type-specific Ps of 3.61±0.39 (w/w) as shown inTable 1, above.

Vaccine Formulation for LPS/Endotoxoids-Conjugates

Enterotoxoid A and Cytotoxoid B conjugates of LPS S. enteritidis, S.dysenteriae and S. paratyphi A were combined. Stoichiometric features ofthe conjugates showed a mean ratio Protein/each of the type-specificLPS/Endotoxoid of 3.61±0.39 (w/w) as shown in Table 1, above.

Combined Broad-Spectrum Enteric Vaccine Formulation for Ps-Conjugatesand LPS (Endotoxoids)-Conjugates Using the Carrier Proteins EnterotoxoidA and Cytotoxoid B

Enterotoxoid A conjugates of PsVi, Ps0139 and PsK1 and Cytotoxoid Bconjugates of LPS (Endotoxoids) S. enteritidis, S. dysenteriae and S.paratyphi A were combined for the purpose.

Dose and Formulations of the Exemplary Vaccines

According to the stoichiometry of the molecular constructs reportedabove in Table 1, the injected dose is ca. 1.0 μg for each Ps/LPS(Endotoxoid) conjugated present in each molecular construct and for eachToxoid (ca. 3.0 μg) contained in the Vaccine Formulation; the dosebecomes ca. 6.0 μg of total protein amount when the Vaccine Formulationcontains the combined Toxoids for the same or different triads ofcarried Ps/LPS (Endotoxoid) antigens (Broad-spectrum Vaccine); AlPO₄ isused as adjuvant at the fixed dose of 0.5 mg/dose (equivalent to ca.0.120 mg of Alum). Adsorption of each multivalent molecular construct tothe mineral adjuvant occurred at ≥80%, on weight basis, as estimated byinhibition-ELISA.

Animals

Each group of animals selected for each of the below reportedimmunization experiments, contained 10 female Balb/c mice.

Route

i. p.

Immunization Schedule

0, 2, 4 weeks; bleeding at week 0, 2, 4, 6.

Control immunization with plain Ps antigens were omitted on the basis ofthe historical knowledge that highly purified Ps antigens are notsignificantly immunogenic in mammalians and do not “boost” IgG isotypeantibodies following repeated injections of it.

ELISA Titers

Titers expressed as end-point reaction showing O.D.≥2.0 relative to thecontrol reactions for each type-specific Ps/LPS (Endotoxoid) and the twoprotein Toxoids. Sera pool dilutions are performed serially, in twofoldfashion, starting from dilution 1/200.

Immunological Results

Geometric Mean Titers of IgG to specific Ps/LPS (Endotoxoid) or to eachof the two Toxoids, in murine sera pool, as determined by ELISA. SD iswithin ±25% of the reported Geometric Mean. Unless otherwise indicated,the statistical significance among sera titers (determined by t-test)was <0.01. Results are summarized in the following Table 3 and 4.

In Vitro Neutralization of the Homologous Toxins

Performed as reported by Porro et al. (1980) for Diphtheria Toxin and asPavliakova et al. (2000) for C. difficile Toxins.

Table 3 illustrates the immunoresponse of mice to the molecular modelinvolving Enterotoxoid A and Cytotoxoid B as carrier protein for Psantigens of E. coli, V. cholerae, S. typhi.

TABLE 3 Enterotoxoid A Cytotoxoid B Ps W 0 W 2 W 4 W 6 W 0 W 2 W 4 W 6Vi <200 200 2,600 15,800 <200 200 2,200 18,900 K1 <200 200 3,200 12,400<200 200 2,400 20,000 0139 <200 200 1,800 11,600 <200 200 1,200 14,800Tox <200 2,800 25,800 84,400 <200 3,200 32,600 95,400

Table 4 shows the immunoresponse of mice to the molecular modelinvolving Enterotoxoid A and Cytotoxoid B as carrier for LPS/Endotoxoidsantigens of S. enteritidis, S. paratyphi A, S. dysenteriae.

TABLE 4 Enterotoxoid A Cytotoxoid B LPS(Endotoxoid) W 0 W 2 W 4 W 6 W 0W 2 W 4 W 6 S. enteritidis <200 400 3,600 12,800 <200 200 1,800 10,400S. paratyphi A <200 200 2,400 14,800 <200 400 3,600 16,400 S.dysenteriae <200 200 2,200 16,400 <200 400 2,800 14,200 Toxoid <2003,400 28,200 66,400 <200 2,400 24,800 84,200

The results depicted in the above Tables 3 and 4 show the anamnesticinduction of biologically functional IgG isotype antibodies for each ofthe four components of the two multivalent molecular constructs(Toxoid-Ps and Toxoid-Endotoxoid multivalent conjugates).

Particularly, any boosting activity on the immune system observed forthe carrier protein is in parallel observed for each of the carried Psantigens, typical and well known behavior of helper T-dependentantigens. The booster effect obtained against the two Toxoids and thebiological activity of the induced anti-Toxoid antibodies also stronglysupports the fact that the multivalent molecular construct has thepotential to work as antigen in humans for the prevention of toxicitydue to the homologous Toxins. The following results were collected,expressed as fold-increase in respect to pre-immunization titers, of thesera GMT obtained following the second booster dose and reported in thefollowing Table 5 as anti-toxic titers.

TABLE 5 Abs to homologous Toxin Toxoid (fold increase for toxinneutralization, in vitro) Enterotoxoid A 456 Cytotoxoid B 562 CRM197 824

The above detailed results, although just focusing on some specificexamples, support the preparation and use of a broad-spectrum entericvaccine for inducing immunity in a mammalian host against the carrierproteins Enterotoxoid A and Cytotoxoid B of C. difficile as well asagainst the carried Ps of E. coli, V. cholerae, S. typhi and the carriedEndotoxoids of S. paratyphi A, S. dysenteriae, S. enteritidis. Based onthe above, the capsular Ps of C. difficile may be also considered as Psantigens carried by the two Toxoids of the homologous pathogen,according to the detailed molecular construct.

The formulation of a broad-spectrum vaccine as the one above reported inExamples 8 and 9, has objective advantages on a vaccine formulationwhich considers the simple and eventual association of each of the sixdifferent Ps/LPS (Endotoxoid) conjugates of each of the two Toxoidproteins:

A) by using the molecular model with built-in multiple-epitopes one mayactually reduce the amount of carrier protein present in thebroad-spectrum formulation (e.g.: the use of just two triads ofconjugates does reduce the amount of protein carrier to ⅓ or 33% of theamount of carrier protein present in the associated formulation of thesix conjugates);B) the number of injections would be reduced to a total of 3 injectionswith an obvious saving of materials and resources in addition to thelower stress of the mammalian host involved (a minimum of 3 injections,one priming dose and two booster doses, for each of the six individualtype-specific vaccines, would result in a total of 18 injections).

BIBLIOGRAPHY

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The invention claimed is:
 1. A vaccine formulation comprising at leastone antigenic multivalent molecular construct consisting of basic unitscomprising the helper-T dependent carrier detoxified proteins selectedbetween Enterotoxoid A and Cytotoxoid B from Clostridium difficilecovalently bound to a minimum of three carbohydrate structures fromenteropathogenic bacteria selected between bacterial polysaccharides ordetoxified lipopolysaccharides of different serological specificity,wherein each carbohydrate structure comprises at least one of therepeating basic epitopes consisting of a minimum of five to twelvemonosaccharide residues, wherein at least one mole of carrier protein isbound to at least one mole of each of the at least three carbohydratestructure or their molar sum to form carried carbohydrate structures ofdifferent serological specificity in a physiologically acceptablevehicle, optionally together with a pharmaceutically acceptable adjuvantor excipient.
 2. A vaccine formulation according to claim 1 wherein saidEnterotoxoid A or Cytotoxoid B originating from Clostridium difficileare detoxified by formalin-treatment or by DNA recombinant technology.3. A vaccine formulation according to claim 1, wherein said carriedcarbohydrate structures of different serological specificity areselected among capsular polysaccharides of Salmonella typhi, Vibriocholerae, Escherichia coli and Clostridium difficile or a combinationthereof.
 4. A vaccine formulation according to claim 1, wherein saiddetoxified lipopolysaccharide is an Endotoxoid.
 5. A vaccine formulationaccording to claim 1 wherein said multivalent molecular construct is,selected between: Enterotoxoid A covalently bound to the capsularpolysaccharides of Salmonella typhi, Vibrio cholerae and Escherichiacoli; Cytotoxoid B covalently bound to the capsular polysaccharides ofSalmonella typhi, Vibrio cholerae and Escherichia coli; Enterotoxoid Acovalently bound to the detoxified lipopolysaccharides/Endotoxoids ofSalmonella enteritidis, Salmonella paratyphi A and Salmonelladysenteriae; Cytotoxoid B covalently bound to the detoxifiedlipopolysaccharides/Endotoxoids of Salmonella enteritidis, Salmonellaparatyphi A and Salmonella dysenteriae.
 6. A vaccine formulationaccording to claim 1 where said vaccine has serological specificity toat least one enteropathogenic bacteria selected from Clostridiumdifficile, Salmonella typhi, Escherichia coli, Vibrio cholerae,Salmonella enteritidis, Shigella flexneri, Shigella sonnei, Salmonellaparatyphi A, Salmonella dysenteriae, Salmonella cholerasuis or acombination thereof.
 7. A vaccine formulation according to claim 1,wherein the dose of each antigenic multivalent molecular constructranges between 0.1 to 100 μg.
 8. A vaccine formulation according toclaim 1, wherein said adjuvant is chosen between a mineral adjuvantselected from aluminium phosphate, aluminium hydroxide; an organicadjuvant selected from squalene-based adjuvants and a biologicaladjuvant selected from monophosphoryl-lipid A and trehalosedicorynomycolate.
 9. A vaccine formulation according to claim 1, whereinthe amount of adjuvant ranges between 0.1-1 mg/dose.
 10. A vaccineformulation according to claim 9, wherein the amount of adjuvant is 0.5mg/dose.
 11. A vaccine formulation according to claim 1, saidformulation being suitable for the administration by subcutaneous,intramuscular, intracutaneous or transcutaneous route.
 12. A method forthe protection of a subject from systemic and enteric infections due toat least one of the enteropathogenic bacteria selected among Clostridiumdifficile, Salmonella typhi, Escherichia coli, Vibrio cholerae,Salmonella enteritidis, Shigella flexneri, Shigella sonnei, Salmonellaparatyphi A, Salmonella dysenteriae, Salmonella cholerasuis, Klebsiella,Enterobacter or a combination thereof, said method comprisingadministering an effective amount of the vaccine of claim
 1. 13. Amethod for the protection of a subject from systemic and entericinfections due to at least one of the enteropathogenic bacteriaaccording to claim 11 where said subject is a human.
 14. A method forthe prevention and/or treatment of viral gastrointestinal infections dueto human noroviruses which comprises administering an effective amountof a vaccine according to claim
 1. 15. A vaccine formulation accordingto claim 1, wherein the dose of each antigenic multivalent molecularconstruct ranges between 1 to 10 μg.
 16. A vaccine formulation accordingto claim 1, wherein said carried carbohydrate structures of differentserological specificity are selected among detoxifiedlipopolysaccharides/Endotoxoids of enteropathogenic bacteria selectedamong Clostridium difficile, Salmonella typhi, Escherichia coli, Vibriocholerae, Salmonella enteritidis, Shigella flexneri, Salmonellaparatyphi A, Salmonella dysenteriae, Shigella sonnei and Salmonellacholerasuis or a combination thereof.
 17. A vaccine formulation asdefined in claim 16 where the enteropathogenic bacteria is Clostridiumdifficile.
 18. A vaccine formulation as defined in claim 16 where theenteropathogenic bacteria is Salmonella typhi.
 19. A vaccine formulationas defined in claim 16 where the enteropathogenic bacteria isEscherichia coli.
 20. A vaccine formulation as defined in claim 16 wherethe enteropathogenic bacteria is Vibrio cholerae.
 21. A vaccineformulation as defined in claim 16 where the enteropathogenic bacteriais Salmonella enteritidis.
 22. A vaccine formulation as defined in claim16 where the enteropathogenic bacteria is Shigella flexneri.
 23. Avaccine formulation as defined in claim 16 where the enteropathogenicbacteria is Salmonella paratyphi A.
 24. A vaccine formulation as definedin claim 16 where the enteropathogenic bacteria is Salmonelladysenteriae.
 25. A vaccine formulation as defined in claim 16 where theenteropathogenic bacteria is Shigella sonnei.
 26. A vaccine formulationas defined in claim 16 where the enteropathogenic bacteria is Salmonellacholerasuis.